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Ecotoxicological information

Toxicity to aquatic algae and cyanobacteria

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Reference
Endpoint:
toxicity to aquatic algae and cyanobacteria
Type of information:
experimental study
Adequacy of study:
key study
Study period:
Experimental starting date 13-5-2014 Experimental completion date 16-5-2014
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
OECD Guideline 201 (Freshwater Alga and Cyanobacteria, Growth Inhibition Test)
Qualifier:
according to guideline
Guideline:
EU Method C.3 (Algal Inhibition test)
GLP compliance:
yes (incl. QA statement)
Specific details on test material used for the study:
The test substance, project sample code T14009 was supplied by the sponsor. Data on handling, stability, composition, purity or other characteristics of the test substance supplied by the sponsor have been used without further verification.
All concentrations given in this report refer to the technical product as supplied by the sponsor.

Stability: Hydrolytically stable (AN 2014)
Water solubility: 815 mg/L
Storage: -25°C
Batch/Lot: 1205442102
Analytical monitoring:
yes
Remarks:
High Performance Liquid Chromatography (HPLC)
Details on sampling:
Based on the results of a range finding study significant inhibition occurred at 1 mg/L. The following final nominal test concentrations were prepared: 0.05, 0.125, 0.312, 0.78, and 1.95 mg/L.

Samples were taken at the start of the test at all concentrations from parallel vessels, and in the control. At the end of the test all test concentrations were sampled from the analytical parallel replicate not containing algae and also from all of the actual test vessels (containing algae) at all concentrations.
At least 10 mL was sampled in each case. Samples from the actual test replicates were filtered using a 0.45 μm filter to remove algae, Filters were primed with the relevant solution before use. Further dilution to within the range of the calibration curve was then carried out.
The samples were analyzed immediately after sampling.
Vehicle:
no
Details on test solutions:
Preparation of the stock solutions
The test substance is sufficiently soluble in water. To prepare the stock solution 0.0118 g of test substance was weighed on an analytical balance and then dissolved directly in approx. 80 mL test medium. The stock solution was agitated mechanically for approximately 45 minutes to completely dissolve the test substance.
The pH of the stock solution was checked and found to be 8.1 and was not adjusted. After this the stock solution was filled up to 100 mL with test medium.
A clear stock solution with a nominal concentration of 118 mg/L was obtained.

Preparation of the test solutions
The test solutions were prepared by addition of the adequate amounts of stock solution (So as to reach desired concentration in 40 ml) to the test vessels, then test medium was added up to the required final volume.
Six control vessels containing only test medium were included in the test.
An additional parallel series with the same concentrations but without addition of inoculum was also prepared for chemical analyses.
Test organisms (species):
Pseudokirchneriella subcapitata (previous names: Raphidocelis subcapitata, Selenastrum capricornutum)
Details on test organisms:
The test was carried out with the freshwater unicellular algae P. subcapitata (CCAP 278/4) obtained from the Culture Collection of Algae and Protozoa SAMS Research Services Ltd. Dunstaffnage Marine Laboratory, Dunbeg, Argyll, Scotland. After purchasing this strain was cultured and maintained. Cultures on sloped agar tubes were stored at 4°C in the dark until required. Exponentially growing cultures are maintained at 23 ± 2°C in a temperature-controlled illuminated orbital incubator and are re-cultured under sterile conditions weekly to keep the algae in this phase.
Test type:
static
Water media type:
freshwater
Limit test:
no
Total exposure duration:
72 h
Remarks on exposure duration:
The extinction of light in each Erlenmeyer flask was measured after 0, 24, 48 and 72 hours.
Test temperature:
23 ± 2°C
pH:
pH 8.1
Nominal and measured concentrations:
The following final nominal test concentrations were prepared: 0.05, 0.125, 0.312, 0.78, and 1.95 mg/L.
Geometric mean measured concentrations: 0.018, 0.031, 0.082, 0.20 and 0.47 mg/L.
Details on test conditions:
De-ionised water:
The de-ionised water used in the study contained less than 10 μg/L of copper (not measured under GLP), with a conductivity of less than 5 μS/cm and less than 2.0 mg/L NPOC-content.

Test vessels:
The test was performed in 100 mL Erlenmeyer flasks containing 40 mL of medium. The test flasks were closed with cotton-wool stoppers.

Culturing cabinet and test conditions:
The test was carried out in a temperature-controlled illuminated orbital incubator, in which the temperature was maintained at 23 ± 2°C, and a continuous uniform illumination was provided in the spectral range of 400 to 700 nm by using fluorescent lamps at a distance of about 0.36 ± 0.02 m from the algal cultures. The light intensity was in the range of 60 to 120 μE·m-2·s-1 (= μmol.m-2·s-1). The test vessels were rotated continuously at a speed sufficient to prevent sedimentation of the algae.

Test medium:
The test medium (OECD algae medium) was prepared according to the OECD and EC guideline (OECD, 2011 and Commission Regulation, 2008) with the following modification: The NaHCO3 concentration of the test medium was increased to 150 mg/L to maintain a more constant pH. The test medium was prepared from concentrated solutions of the mineral salts prepared in deionized water.

Test conditions:
Based on the results of a range finding study significant inhibition occurred at 1 mg/L. The following final nominal test concentrations were prepared: 0.05, 0.125, 0.312, 0.78, and 1.95 mg/L.

Preparation of the inoculum:
The initial stock culture was inoculated with P. subcapitata from a sloped agar tube and checked for purity by microscopic means. This algal stock culture (40 mL) of P. subcapitata was regularly transferred to fresh medium to act as inoculum for testing.
The extinction of an exponentially growing stock culture was measured. The cell density was determined using the calibration curve. From this algal culture a dilution was prepared to obtain an initial cell density of approximately 1×10^4 cells/mL in the test medium.

Test procedures:
Culture medium was prepared by diluting the stock mineral salts in an appropriate vessel. This medium was sterilized by filter sterilization (0.2 μm). Adequate amounts of test substance stock solution were added. The Erlenmeyer flasks were then filled with test solution up to a total volume of 40 mL using a sterilized dispenser pipette. Then the inoculum was added to the vessel from an exponentially growing culture. All test concentrations were tested in triplicate. In addition, six replicates of the control were included.
The extinction of light in each Erlenmeyer flask was measured after 0, 24, 48 and 72 hours. Algal medium was used as a blank in the spectrophotometer.

Determination of cell concentrations:
Cell concentrations were determined photometrically with a UV/VIS Spectrophotometer. Measurements were carried out at 436 nm in a cuvette with a light path of 4 cm. To establish the relation between extinction of light and cell density, a calibration curve was made which is checked yearly. From the relation between extinction (E) and counted cell number per mL (N) the following calibration curve was determined using linear regression:
N = (E-0.0386)/4.10^-7
The calibration curve was used to determine the cell density of the inoculum before and during the test when needed.
Reference substance (positive control):
yes
Remarks:
Potassium dichromate
Duration:
72 h
Dose descriptor:
EC10
Effect conc.:
0.052 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
growth rate
Duration:
72 h
Dose descriptor:
EC50
Effect conc.:
0.47 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
growth rate
Duration:
72 h
Dose descriptor:
NOEC
Effect conc.:
0.031 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
growth rate
Duration:
72 h
Dose descriptor:
LOEC
Effect conc.:
0.082 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
growth rate
Duration:
72 h
Dose descriptor:
EC50
Effect conc.:
0.098 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
biomass
Details on results:
The results reported are based on geometric measured mean substance concentrations. All reported concentrations refer to the test substance as received and are thus not corrected for active ingredient content.
No significant effect was observed on growth rate up to 0.031 mg/L. The NOEC is therefore 0.031 mg/L and LOEC is 0.082 mg/L. The ErC10 is 0.052 mg/L and the ErC50 is 0.47 mg/L, confidence limits could not be calculated for rate endpoints. The EbC50 was found to be 0.098 mg/L, with 95% confidence limits of 0.048 and 0.211 mg/L.

Water quality and purity of algae

The pH measurements show a maximum increase of 1.2 pH units.

The temperature varied from 22.3 to 23.1 °C during the test.

Light intensity was 99.8 and 94.4 μmol·m-2·s-1 at the beginning and end of the test, respectively.

All measurements are in accordance with the required conditions described in the study plan.

Five random samples of the control were all pure and not contaminated with bacteria.

Analytical results

The method used met all validity requirements.

The concentrations measured at the start and the end of the test were not within 80 to 120% of the nominal concentrations. Therefore, geometric measured mean concentrations were used for endpoint

calculations. Where recovery was <LOQ ½ of the quantification limit was used.

Quality criteria

The following quality criteria have been met in the present study:

· The cell density of the controls increased at least a factor 16 within 72 hours.

· The mean coefficient of variation for section-by-section specific growth rates in the control cultures must not exceed 35%.

· The coefficient of variation of average specific growth rates during the whole test period in the replicate control cultures should not exceed 7%.

· Analytical quality criteria were met.

· Algae strain used was of acceptable sensitivity.

Validity criteria fulfilled:
yes
Conclusions:
All of the biological and analytical quality criteria were met and the study data can be considered to be an accurate representation of the effects of the test material to fresh water algae. The stability as indicated in the hydrolysis test and the thermal stability as indicated from the sponsor suggested much better stability than was measured during this study. Furthermore the parallel replicates without algae did not appear to give increased recovery. Lack of stability may therefore be due to the effects the slightly elevated temperature in the test 23ºC had on the hydrolysis speed. (12ºC was used in the hydrolysis study) It is also documented (Buback 2000) that peroxides can also be degraded by the influence of light making maintenance of concentrations in algae tests impossible. Considering the high level of illumination during the study this could also be a potential explanation for the loss of the test substance. In reality a high initial concentration is therefore likely the cause of the toxic effect and use of the geometric mean concentrations for endpoint expression should be considered extreme worst case.
Executive summary:

In order to predict the effects of the test chemical in an aquatic environment, the toxicity of Tert-amylperoxy pivalate to freshwater algae was determined using the Algal Growth Inhibition Test in accordance with OECD, EC and ISO test guidelines and with the OECD Principles of Good Laboratory Practice. Slight modifications to the guideline were applied to ensure good growth and pH control of the

cultures.

The toxicity of the test substance to an exponentially growing culture of P. subcapitata was determined over an exposure period of 72 hours. All endpoints are based on geometric mean measured

concentrations.

No significant effect was observed on growth rate up to 0.031 mg/L. The NOEC is therefore 0.031 mg/L and LOEC is 0.082 mg/L. The ErC10 is 0.052 mg/L and the ErC50 is 0.47 mg/L, confidence limits could not be calculated for rate endpoints. The EbC50 was found to be 0.098 mg/L, with 95% confidence limits of 0.048 and 0.211 mg/L.

The test is valid as shown by:

The increase of the extinction of the control over 72 h by a factor of 161

The mean coefficient of variation for section-by-section specific growth rates in the control cultures did not exceed 35%.

The coefficient of variation of average specific growth rates during the whole test period in the replicate control cultures did not exceed 7%.

The set quality criteria for chemical analysis were all met.

The most recent reference test for the algae species tested demonstrated acceptable sensitivity according to the test guideline.

In addition, pH variation in the control did not vary more than 1.5 pH units as recommended in the study guideline.

The results of the chemical analyses show that the test substance did not remain stable in the test system. However sufficient quantification was possible to allow calculations of geometric means of the

measured concentrations as is acceptable in the study guideline.

Description of key information

One valid study perforemd with the freshwater alga P. subcapitata is available. The study was performed according to OECD 201 under GLP.

The ErC10 is 0.052 mg/L and the ErC50 is 0.47 mg/L.

Key value for chemical safety assessment

EC50 for freshwater algae:
0.47 mg/L
EC10 or NOEC for freshwater algae:
0.052 mg/L

Additional information